PROJECT SUMMARY Epilepsy, characterized by recurrent spontaneous seizures, affects over 50 million people worldwide and is one of the most common neurological disorders. Mutations in the voltage-gated sodium channel gene, SCN8A, have recently been identified as an important cause of severe pediatric epilepsy. With the advent of next- generation sequencing, over 100 pathogenic SCN8A mutations have been identified in patients. 15 of these mutations are located in the S4 voltage-sensor domains that are critical for voltage-dependent gating of the SCN8A channel protein, Nav1.6. Notably, patients with these mutations exhibit a strikingly broad spectrum of clinical phenotypes, including intellectual disability, ataxic gait, hypotonia, and treatment-resistant epilepsy. To better understand how mutations in the voltage-sensor domains can cause this broad range of clinical phenotypes, we utilized CRISPR/Cas9 to generate mice with mutations in the Scn8a DIIS4 voltage-sensor domain. We ultimately generated two distinct mouse lines with novel, in-frame Scn8a mutations: a 9bp deletion that removes three amino acid residues (R850_F852del, denoted ?9bp) and a 4bp substitution that alters two amino acid residues (R850L+V851S, denoted RV>LS). Mice that are homozygous for either mutation exhibit distinct movement abnormalities, reduced size, and shortened lifespans. However, the severity of these phenotypes varies between the two mouse lines, suggesting that each mutation has a unique effect on Nav1.6 function. My preliminary data demonstrates that heterozygotes for the Scn8a ?9bp mutation have increased resistance to flurothyl-induced seizures, indicating that these mutations also impact seizure profiles. Taking into account these preliminary data, I hypothesize that mice expressing either the Scn8a ?9bp or RV>LS mutation will demonstrate altered seizure profiles, behavior abnormalities, and motor function. In Aim 1, I will establish the effect of each mutation on susceptibility to induced seizures. Mice expressing each mutation will also be monitored for spontaneous seizures using EEG analysis. In Aim 2, I will determine whether either Scn8a mutation impacts mouse behavior, motor coordination, or motor neuron function. Together, these two Aims will further our understanding of how altered SCN8A S4 voltage-sensor function contributes to the phenotypes observed in patients with SCN8A mutations. Our long-term goal is to use these mouse models to examine how SCN8A dysfunction causes epilepsy, and evaluate potential treatments for patients with SCN8A- derived epilepsy.